Diseases caused by Trypanosoma brucei spp. represent health hazards for many disadvantaged countries. Trypanosomatids diverged early in evolution and possess unique RNA processing pathways, such as mitochondrial internal U-insertion/deletion mRNA editing and ubiquitous 3? end uridylation. The principal role of uridylation in mitochondrial RNA metabolism enthused identification of the first terminal uridyltransferase (TUTase) RET1. Progressing studies of TUTases uncovered their wide phylogenetic distribution, diverse structures and profound transcriptome-shaping roles in eukaryotic cells. In this proposal, we expand earlier finding of RET1 targeting all RNA classes into a mechanistic investigation of uridylation-driven RNA processing and decay. In preliminary studies, we discovered that RET1 TUTase, DSS1 3?-5? exonuclease and three proteins lacking recognizable motifs constitute a core processing complex termed the mitochondrial 3? processome (MPsome). We show that uridylation-targeted degradation most likely operates in concert with antisense transcription to define 3? boundaries of mature guide RNAs. Finally, we determined a high-resolution crystal structure of RET1 with bound substrates. The proposed experiments will resolve the long-standing questions of how primary mitochondrial transcripts are processed prior to entering editing pathway and how uridylation controls the abundance of translationally-competent mRNAs. Aim 1 will define the molecular architecture of the MPsome. A hybrid structural biology approach will include chemical crosslinking-assisted mass spectrometry (XL-MS) and cryoelectron microscopy (cryoEM). We will build a near-atomic resolution complex model with bound RNA substrates. Aim 2 will determine the mechanism by which 3? uridylation targets guide RNAs for processing. We propose that uridylation-coupled, antisense transcription-controlled 3?-5? RNA degradation represents the major processing pathway of primary transcripts and will focus on gRNA biogenesis. We will identify in vivo MPsome binding sites, map positions of primary transcripts and reconstitute processing reactions in vitro. Aim 3 will investigate the relationship between mRNA 5? modification and 3?5? degradation. The 5? termini of mature maxicircle transcripts (rRNAs and mRNAs) are monophosphorylated while minicircle-encoded gRNAs retain triphosphates characteristic of transcription start site. We present evidence that NUDIX hydrolase MERS1 recognizes 5? termini of primary maxicircle transcripts to remove pyrophosphate and propose a mechanism linking mRNA's 5? modification and 3?-5? exonucleolytic decay. This Aim will investigate the specificty of MERS1 targeting and functional interactions with the MPsome. Aim 4 will develop small-molecule inhibitors of RET1 by molecular dynamics-assisted screening of virtual libraries. We will apply chemical probes to complement genetic approaches developed in Aims 2 and 3. Although this Aim's main objective is to search for chemical knockdown tools, the benefits of targeting a multifunctional enzyme are apparent, and the likelihood of converting such advances into future therapies is high.